Genetically modified non-human animal with human or chimeric OX40

Abstract

The present disclosure relates to the genetically modified non-human animals that express a human or chimeric OX40, and methods of use thereof.

What is claimed is: 
     
       1. A method for making a genetically-modified mouse, comprising:
 (1) replacing in a fertilized egg or an embryonic stem cell, at an endogenous OX40 gene locus, a nucleic acid sequence encoding a portion of the extracellular region of an endogenous OX40 protein with a nucleic acid sequence encoding a portion of an extracellular region of a human OX40 protein, wherein the replaced nucleic acid sequence comprises a portion of exon 1, exons 2-4 and a portion of exon 5 of the endogenous OX40 gene; 
 (2) transplanting the fertilized egg obtained in step (1) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (1) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to obtain a child mouse; and 
 (3) mating the child mouse obtained in step (2) to obtain a homozygote mouse, 
 wherein the homozygote mouse detectably expresses a humanized OX40 protein comprising a humanized OX40 extracellular region and an endogenous OX40 cytoplasmic region on the surface of activated T cells, wherein the humanized OX40 protein comprises SEQ ID NO: 32. 
 
     
     
       2. The method of  claim 1 , wherein the sequence encoding the humanized OX40 protein is operably linked to an endogenous regulatory element at the endogenous OX40 gene locus. 
     
     
       3. The method of  claim 1 , wherein the genetically-modified mouse does not express the endogenous OX40 protein. 
     
     
       4. The method of  claim 1 , wherein an anti-human OX40 antibody can bind to the humanized OX40 protein expressed in the mouse and increase immune response in the mouse. 
     
     
       5. The method of  claim 1 , wherein the mouse can be used to test effectiveness of an anti-human OX40 antibody for treating cancer. 
     
     
       6. A method for making a genetically-modified mouse, comprising:
 (1) providing a plasmid comprising a human OX40 gene fragment, flanked by a 5′ homology arm and a 3′ homology arm, wherein the 5′ and 3′ homology arms target an endogenous OX40 gene; 
 (2) providing two small guide RNAs (sgRNAs) that target the endogenous OX40 gene; 
 (3) modifying genome of a fertilized egg or an embryonic stem cell by using the plasmid of step (1), the sgRNAs of step (2), and Cas9; and 
 (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudopregnant female mouse or transplanting the embryonic stem cell obtained in step (3) into a blastocyst which is then transplanted into the oviduct of a pseudopregnant female mouse to produce a child mouse that functionally expresses a humanized OX40 protein, 
 (5) mating the child mouse obtained in step (2) to obtain a homozygote mouse, 
 wherein the fertilized egg is modified by CRISPR with sgRNAs that target a 5′-terminal targeting site selected from the group consisting of SEQ ID NOS: 1-7 and a 3′-terminal targeting site selected from the group consisting of SEQ ID NOS: 8-15, and the humanized OX40 protein comprises SEQ ID NO: 32. 
 
     
     
       7. The method of  claim 6 , wherein the 5′-terminal targeting site is SEQ ID NO: 1 and the 3′-terminal targeting site is SEQ ID NO: 11. 
     
     
       8. The method of  claim 6 , wherein the sequence encoding the humanized OX40 protein is operably linked to an endogenous regulatory element at the endogenous OX40 gene locus. 
     
     
       9. The method of  claim 6 , wherein the genetically-modified mouse does not express an endogenous OX40 protein. 
     
     
       10. The method of  claim 6 , wherein an anti-human OX40 antibody can bind to the humanized OX40 protein expressed in the mouse and increase immune response in the mouse. 
     
     
       11. The method of  claim 6 , wherein the mouse can be used to test effectiveness of an anti-human OX40 antibody for treating cancer.